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KERAGAMAN GENETIK BEBERAPAKLON DURIAN(Durio Berita Biologi 10(3) - Desember 2010 KERAGAMAN GENETIK BEBERAPAKLON DURIAN (Durio zibethinus Murray) AS AL JAWA BARAT BERDASARKAN SIDIK RANDOM AMPLIFIED POLIMORPHIC DNA1 [Genetic Diversity of Durian (Durio zibethinus Murray) from West Java Based on Random Amplified Polymorphic DNA Fingerprint] Kusumadewi Sri Yulita213* dan Muna Murnianjari3 2Pusat Penelitian Biologi-LIPI, Raya Bogor Km. 46, Cibinong 16911 3FMIPA Universitas Negeri Jakarta •email: [email protected] ABSTRACT Durian (Durio zibethinus) is one of the most popular tropical fruits in SE Asia. Indonesia has several local clones that have not yet been widely introduced to local fruit markets. This present study aimed to assess genetic diversity of 17 clones of durian from West Java based on RAPD fingerprints. Thirty RAPD's primers were initially screened and four were selected for the analysis. These four primers (OPA 13, OPD 8, OPN 6 and OPA 18) generated 63 scorable bands to which 100% of them were polymorphic. OPA-13 at 700bp was exclusively possessed by Tambleg clone and other bands were shared among the other clones. Clustering analysis was performed based on RAPD profiles using the UPGMA method. The range of genetic similarity value among genotypes was 0.15-0.73 suggesting high genetic variation among them. Results from genetic diversity analysis based on plant propagation system showed a higher genetic diversity value in occulating (87.30%) plants than that of grafting (60.32%). Kata kunci/key word: Keragaman genetik/genetic diversity, durian (Durio zibethinus), RAPD, klon/clone. PENDAHULUAN merupakan salah satu penanda molekuler yang umum Durio zibethinus Murray merupakan salah satu digunakan untuk melihat polimorfisme di tingkat DNA komoditas buah-buahan yang banyak dibudidayakan (Chakrabarti et al., 2001) dengan teknik PCR saat ini dan bernilai ekonomi cukup penting. Beberapa (Polymerase Chain Reaction). Penggunaan penanda kultivar durian unggul yang telah dikembangkan di RAPD mudah dalam persiapannya, relatif sederhana Indonesia antara lain Sunan, Sukun, Petruk, Sitokong, namun memberikan hasil yang lebih cepat Mas, Otong, Tembaga, Perwira, Bokor, Hepe dan Sriwig dibandingkan penanda molekuler lainnya, tidak (Subhadrabandhu et al, 1997). Besarnya keragaman membutuhkan pengetahuan mengenai urutan DNA kultivar merupakan sumber plasma nutfah penting dari organisme target (Hillis et al., 1996), jumlah DNA sebagai bahan seleksi untuk pemuliaan tanaman durian. yang digunakan dalam reaksi hanya sedikit (± 25 Di wilayah Jawa Barat terdapat beberapa klon durian nanogram per reaksi), penggunaan peralatan yang telah dikenal masyarakat karena sudah lama laboratorium yang minimal, non-radioaktif, dan dapat dibudidayakan dan pada umumnya merupakan buah menggunakan primer universal yang sudah ada (Soltis yang diminati. et al., 1998). Pada saat ini, penanda RAPD telah banyak Saat ini, penelitian tentang keragaman genetik digunakan dalam penelitian keragaman genetik beberapa klon durian asal Jawa Barat belum dilakukan, (Adetula, 2006; Fan et al., 2004, Guo et al, 2007; Ishak, padahal durian merupakan salah satu kekayaan plasma 2000; Jain et al, 2007; Nurhaimi-Haris et al, 1998; nutfah buah-buahan yang penting untuk Poerba et al., 2007; Toruan-Mathius et al, 2002, Ferriol dikembangkan. Salah satu cara untuk mengetahui et al, 2003), sidik DNA (Baum, 1999; Chakrabarti et al, keragaman plasma nutfah durian adalah dengan melihat 2001; Chakrabarti et al, 2006; Zacarias et al, 2004) dan keragaman genetik. Keragaman genetik dapat identitas kultivar (Galderisi et al, 1999; Keller- diestimasi melalui perbedaan individu pada berbagai Przybyflcowicz et al., 2006). tingkatan misalnya pada tingkat DNA baik berupa Penelitian kali ini bertujuan untuk mengetahui perbedaan urutan maupun ukuran basa nukleotida. keragaman genetik antar klon durian asal Jawa Barat RAPD {Random Amplified Polymorphic DNA) dan memperoleh identitas klon durian berdasarkan 'Diterima: 23 Agustus 2009 - Disetujui: 18 Februari 2010 269 Yulita dan Murnianjari - Keragaman Genetik Klon Durian Berdasarkan Sidik RAPD Tabel 1. Daflar sampel durian Tahun No. Nama klon Material Asal Tanam 1. Tambleg 1 (Tl) Okulasi Pandeglang 1998 2. Tambleg 2 (T2) Okulasi Pandeglang 1998 3. Si Lilin 1 (Ll)^ Enten Ragunan 1983 4. Isi Manis (IM) Okulasi Pandeglang 1998 5. Sibadak 1 (SB1) Okulasi Sumedang 1998 6. Simaung (SM) Okulasi Pandeglang 1998 7. Kendil (KE) Okulasi Cibinong 1998 8. Sitokong KJ 1 (ST1) Enten Rramat Jati 1998 9. Handalam (HA) Enten Jakarta 1986 10. Nian 1 (Nl) Enten Pandeglang 1998 11. Kamarung (KA) Enten Parung 1998 12. Bokor 1 (Bl) enten Cipaku 1986 13. Si Pingku (SP) Enten Rancamaya 1986 14. Bokor 2 (B2) Okulasi Sumedang 1998 15. Sibadak 2 (SB2) Enten Sukaraja 1986 16. Sitokong KJ 3 (ST3) Enten Kramat Jati 1986 17 Si Lilin 2 (L2) Enten Rancamaya 1986 (36°C selama 1 menit) dan fase pemanjangan (72° C profil pita RAPD. selama 2 menit) (Williams etal, 1990). Setelah45 siklus BAHANDANMETODE selesai, proses amplifikasi PCR diakhiri dengan fase Sampel tanaman durian yang dianalisis sejumlah pemanjangan pada suhu 72° C selama 5 menit. 17 yang berasal dari Kebun Plasma Nutfah (KPN) LIPI, Perkiraan keragaman genetika berdasarkan analisis Cibinong dan dikoleksi dalam bentuk daun yang pengelompokan dan ordinasi dikeringkan dalam silica gel (Tabel 1). Setiap pita RAPD dianggap sebagai satu lokus Ekstraksi total DNA genom putatif. Hanya lokus yang menunjukkan pita yang jelas Total DNA genom diisolasi dengan yang digunakan untuk diskor 1 bila ada pita dan 0 bila menggunakan protokol CTAB (Delaporta et al, 1983) tidak ada pita. Data RAPD yang telah diskor yang dimodifikasi dengan penambahan RNAse 200 \ig/ selanjutnya digunakan untuk menghitung persentase mL. Lima uL total DNA genom di elektrophoresis dalam lokus polimorfik (P). 0.7% gel agarose dalam larutan penyangga TAE, Data skoring dikelompokkan hingga membentuk kemudian diwarnai dengan ethidium bromida dan difoto matriks binari di program Microsoft Excel. Matriks menggunakan lampu ultraviolet untuk memastikan tersebut kemudian diolah menggunakan program kuantitas dan kualitas DNA. DNA yang telah diisolasi SIMQUAL (Similarity for qualitative data) dengan disimpan dalam -20°C. koefisien DICE dalam paket NTSYS-pc versi 2.02 (Rohlf, AnalisisRAPD 1993). Hasil analisis yang dihasilkan berupa Skrining dilakukanterhadap 30 primer A, C dan dendrogram pengelompokan berdasarkan UPGMA N (Operon Technologies) untuk memperoleh primer (Unweightedpair group with arithmetical averages). dengan tingkat keberulangan tinggi dan konsisten Matriks kesamaan dari data ini digunakan untuk digunakan dalam analisis RAPD. Volume reaksi kembali untuk memperoleh ordinasi 3 dimensi total PCR adalah 15 ul yang terdiri atas 1 x PCR Master berdasarkan nilai eigen dan vektor eigen menggunakan Mix (Fermentas), 2 uM primer (Promega), dan ~10 ng program NTSYS-pc 2.02 (Rohlf, 1993). DNA template. Kondisi mesin PCR untuk amplifikasi Perkiraan keragaman genetik berdasarkan cara DNA diawali dengan denaturasi awal pada suhu 94°C perbanyakan tanaman selama 2 menit, diikuti oleh 45 siklus yang terdiri dari: Indeks keragaman genetik untuk memperkirakan fase denaturasi (94°C selama 1 menit); fase penempelan keragaman genetik berdasarkan cara perbanyakan 270 Berita Biologi 10(3) - Desember 2010 Tabel 2. Urutan primer dan jumlah fragmen DNAyang terbentuk Jumlah ftagmen DNA Fragmen DNA Primer Urutan DNA yang terbentuk polimorfik OPA-13 CAGCACCCAC 18 18 (100%) OPA-18 AGGTGACCGT 11 11(100%) OPN-6 GAGACGCACA 20 20 (100%) OPD-8 GTGTGCCCCA 14 14 (100%) Jumlah 63 63 (100%) Tabel 3. Matriks kesamaan genetik antar 17 klon durian asal Jawa Barait Tl T2 LI IM SB1 ST1 HA K SM KE Nl Bl SP B2 SB2 ST3 L2 Tl 1.00 T2 0.24 1.00 LI 0.16 0.20 1.00 IM 0.15 0.26 0.45 1.00 SB1 0.33 0.21 0.41 0.20 1.00 ST1 0.29 0.31 0.62 0.30 0.40 1.00 HA 0.35 0.36 0.50 0.34 0.37 0.67 1.00 K 0.27 0.44 0.52 0.36 0.38 0.61 0.72 1.00 SM 0.25 0.22 0.27 0.37 0.22 0.30 0.29 0.29 1.00 KE 0.40 0.30 0.35 0.49 0.41 0.33 0.42 0.49 0.34 1.00 Nl 0.26 0.28 0.33 0.41 0.38 0.36 0.39 0.45 0.56 0.64 1.00 Bl 0.18 0.22 0.59 0.36 0.38 0.61 0.72 0.58 0.24 0.32 0.40 1.00 SP 0.36 0.30 0.44 0.29 0.46 0.52 0.56 0.58 0.29 0.32 0.40 0.58 1.00 B2 0.30 0.16 0.32 0.31 0.42 0.29 0.43 0.36 0.19 0.40 0.37 0.45 0.36 1.00 SB2 0.20 0.24 0.64 0.38 0.42 0.57 0.61 0.73 0.25 0.34 0.42 0.73 0.55 0.40 1.00 ST3 0.25 0.41 0.55 0.40 0.50 0.56 0.52 0.62 0.33 0.46 0.43 0.54 0.54 0.42 0.67 1.00 L2 0.22 0.17 0.52 0.25 0.36 0.53 0.57 0.50 0.27 0.24 0.33 0.60 0.70 0.33 0.67 0.64 1.00 tanaman (okulasi dan enten), yaitu berdasarkan indeks dibanding tiga primer lainnya. keragaman Nei dan Shannon dengan menggunakan Analisis pengelompokan antar klon durian program POPGENE 32 versi 1.31 (Yehetal., 1997). Berdasarkan matriks kesamaan genetik, nilai kesamaan genetik 17 klon durian berkisar antara 0.15- HAS1L 0.73 (Tabel 3). Semakin tinggi nilai jarak genetik maka ProfilpitaRAPD tingkat kesamaan semakin besar dan sebaliknya. Nilai Enam tanaman durian digunakan dalam proses jarak genetik terbesar (0.73) terdapat antaraK-SB2 dan skrining primer untuk memilih primer yang B1-SB2. Nilai j arak genetik terkecil yaitu 0.15 terdapat menghasilkan pita DNA polimorfik. Hasil seleksi antara klon Tl-IM. skrining menggunakan 30 primer menunjukkan bahwa Analisis kluster menunjukkan pemisahan primerOPA 13, OPA18, OPN6 danOPD 8 menghasilkan tanaman durian ke dalam kluster yang mengelompok profil pita RAPD yang jelas dan mudah dibaca.
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